Angiotensin II (Ang II)—the octapeptide (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe)—is a pleiotropic vasoactive peptide that binds to two distinct receptors: the Ang II type 1 (AT1)and type 2 (AT2) receptors. Activation of the renin-angiotensin-aldostrone system (RAAS) results in vascular hypertrophy, vasoconstriction, salt and water retention, and hypertension. These effects are mediated predominantly by AT1 receptors. Other Ang II-mediated effects, including cell death, vasodilation, and natriuresis, are mediated by AT2 receptor activation. The understanding of Ang II signalling mechanisms remains incomplete. AT1 receptor activation triggers a variety of intracellular systems, including tyrosine kinase-induced protein phosphorylation, production of arachidonic acid metabolites, alteration of reactive oxidant species activities, and fluxes in intracellular Ca2+ concentrations. AT2 receptor activation leads to stimulation of bradykinin, nitric oxide production, and prostaglandin metabolism, which are, in large part, opposite to the effects of the AT1 receptor. (See: Berry C, Touyz R, Dominiczak A F, Webb R C, Johns D G.: Am J Physiol Heart Circ Physiol. 2001 December; 281 (6):H2337-65. Angiotensin receptors: signalling, vascular pathophysiology, and interactions with ceramide).
Ang II is the active component of the renin-angiotensin-aldosterone system (RAAS). It plays an important physiological role in the regulation of blood pressure, plasma volume, sympathetic nervous activity, and thirst responses. Ang II also has a pathophysiological role in cardiac hypertrophy, myocardial infarction, hypertension, chronic obstructive pulmonary disease, liver fibrosis and atherosclerosis. It is produced systemically via the classical RAAS and locally via tissue RAAS. In the classical RAAS, circulating renal-derived renin cleaves hepatic-derived angiotensinogen to form the decapeptide Ang I, which is converted by ACE in the lungs to the active Ang II. Ang I can also be processed into the heptapeptide Ang-(1-7) by tissue endopeptidases.
The RAAS system is illustrated schematically in FIG. 1 hereto which is based on FIG. 1 in the article by Foote et al. in Ann. Pharmacother. 27: 1495-1503 (1993).
In addition to the RAAS playing an important role in the normal cardiovascular homeostasis, over activity of the RAAS has been implicated in the development of various cardiovascular diseases, such as hypertension, congestive heart failure, coronary ischemia and renal insufficiency. After myocardial infarction (MI), RAAS becomes activated. Specifically the AT1 receptor seems to play a prominent role in post-MI remodelling, since AT1 receptor expression is increased after MI and in left ventricular dysfunction. Therefore drugs that interfere with RAAS, such as ACE inhibitors and AT1 receptor antagonists have been shown to be of great therapeutic benefit in the treatment of such cardiovascular disorders.
The anatomic coincidence between the expression of ACE and the AT1 receptors with normal and pathological expressions of collagen formation is evident. High-density ACE and Ang II receptor binding are markers of active collagen turnover. Alfa-SMA-containing, fibroblast-like cells, found within valve leaflets, adventitia, and various sites of fibrous tissue formation, express genes encoding for ACE, AT1 receptors, and fibrillar collagens. Myofibroblasts, MyoFbs, could therefore be considered a “metabolic entity” regulating their own collagen turnover.
ACE binding density is related primary to the presence of myoFbs. The disappearance of ACE-positive cells or a reduction in their absolute number would reduce ACE binding density at sites of fibrosis. Such is the case with old sarcoid granulomas. Both ACE and Ang II receptor binding densities in the infracted rat heart remain high for many months after the MI, as does ACE activity. Each is in the keeping with the persistence of myoFbs at the infracted site. (Weber, K. T.: Extracellular Matrix Remodelling in Heart Failure: A Role for De Novo Angiotensin II Generation. Circulation, Volume 96(11), Dec. 2, 1997, 4065-4082.)
For heart, kidneys, lungs and liver alike, fibrosis represents a common pathway to their failure. Understanding pathophysiologic mechanisms involved in organ fibrosis are therefore of considerable interest, particularly given the potential for protective pharmacological strategies. Tissue repair involves inflammatory cells, including members of the monocyte/macrophage lineage, integral to initiating the repair process; and myofibroblasts, phenotypically transformed interstitial fibroblasts, responsible for collagen turnover and fibrous tissue formation. Each of these cellular events in the microenvironment of repair are associated with molecular events that lead to the de novo generation of Ang II. In an autocrine/paracrine manner, this peptide regulates expression of Transforming growth factor beta 1, TGF-beta 1, via angiotensin (AT1) receptor-ligand binding. It is this cytokine that contributes to phenotypic conversion of fibroblasts to myofibroblasts (myoFb) and regulates myofibroblast turnover of collagen. ACE inhibition or AT1 receptor antagonisms each prevent many of these molecular and cellular responses that eventuate in fibrosis and therefore have been found to be protective interventions. (See: Weber K T. Fibrosis, a common pathway to organ failure: angiotensin II and tissue repair. Semin Nephrol. 1997 September; 17(5):467-91 and references therein).
Ang II may regulate tissue fibrosis via the activation of mesenchymal cells. For example, Ang II stimulates the proliferation of cardiac fibroblasts in vitro via activation of AT1. The presence of AT1 receptors has also been demonstrated on cardiac fibroblasts in vitro. Most of the profibrotic effects of Ang II appear to be mediated via this receptor; however, increased AT2 expression on cardiac fibroblasts has been detected in hypertrophied human heart, and the balance between the expression of these two subtypes may be critical in determining the response to Ang II. (See: Am. J. Respir. Crit. Care Med., Volume 161, Number 6, June 2000, 1999-2004 Angiotensin II Is Mitogenic for Human Lung Fibroblasts via Activation of the Type 1 Receptor Richard P. Marshall, Robin J. McAnulty, and Geoffrey J. Laurent and references therein).
The Ang II receptors can be distinguished according to inhibition by specific antagonists. AT1 receptors are selectively antagonized by biphenylimidazoles, such as Losartan, whereas tetrahydroimidazopyridines specifically inhibit AT2 receptors. The AT2 receptor may also be selectively activated by CGP-42112A. This is a hexapeptide analog of Ang II, which may also inhibit the AT2 receptor, depending on concentration). Two other angiotensin receptors have been described: AT3 and AT4 subtypes.
In rodents, the AT1 receptor has two functionally distinct subtypes, AT1A and AT1B, with >95% amino acid sequence homology.
The second major angiotensin receptor isoform is the AT2 receptor. It has low amino acid sequence homology (˜34%) with AT1A or AT1B receptors. Although the exact signaling pathways and the functional roles of AT2 receptors are unclear, these receptors may antagonize, under physiological conditions, AT1-mediated actions inhibiting cell growth and by inducing apoptosis and vasodilation. The exact role of AT2 receptors in cardiovascular disease remains to be defined.
Other receptors for Ang II besides AT1 and AT2 are known and are generally referred to as ATatypical (see Kang et al., Am. Heart J. 127: 1388-1401 (1994)).
The suppression of Ang II's effects has been used therapeutically, for example in the management of hypertension and heart failure. This has been achieved in a number of ways: by the use of renin inhibitors which block the conversion of angiotensinogen to Ang I (the precursor to Ang II); by the use of angiotensin converting enzyme inhibitors (ACE-I) that block the conversion of Ang I to Ang II (and also block bioconversion of bradykinin and prostaglandins); by the use of anti-Ang II-antibodies; and by the use of Ang II-receptor antagonists.
Beta blockers are most commonly used in treatment of arrhythmias. Anti-arrhythmic drugs have had limited overall success and calcium channel blockers can sometimes induce arrhythmias. No single agent shows superiority, with the possible exception of amiodarone. Short-term anti-arrhythmic benefit has been found to be offset by, depending on the specific drug, neutral or negative effects on mortality (Sanguinetti M C and Bennett, P B: Anti-arrhythmic drug target choices and screening. Circulation 2003, 93(6): 491-9257-263). Clearly better anti-arrhythmic drugs are needed.
A publication in Lancet (Lindholm, L H et al. Effect of Losartan on sudden cardiac death in people with diabetes: data from the LIFE study, The Lancet, 2003, 362: 619-620) revealed that AT1 receptor antagonists in addition of being generally favourable to patients with CHF, also reduce the incidence of sudden cardiac death. There exist a few studies showing that AT1 antagonists have an anti-arrhythmia effect on arrhythmias induced by myocardial infarct or in reperfusion after ligation of LAD (Harada K et al. Angiotensin II Type 1a Receptor is involved in the occurrence of reperfusion arrhythmias, Circulation, 1998, 97:315-317, Ozer M K et al. Effects of Captopril and Losartan on myocardial ischemia-reperfusion induced arrhythmias and necrosis in rats, Pharmacological research, 2002, 45 (4), 257-263, Lynch J J et al. EXP3174, The AII antagonist human metabolite of Losartan, but not Losartan nor the Angiotensin-converting enzyme inhibitor captopril, prevents the development of lethal ischemic arrhythmias in a canine model of recent myocardial infarction, JACC, 1999, 34 876-884).
Ang II may be turned into potent antagonists or partial antagonists by changes in their amino acid composition. For instance substituting phenylalanine in position 8 with isoleucine and aspartic acid in position 1 with sarcosine changes the peptide into a potent antagonist.
The specificity towards the AT1 receptor may be increased by cyclisation or bridging of the amino acids in position 3 and 5. Similarly introducing sarcosine in position 1 and glycine in position 8 makes the peptide into a AT1 selective antagonist (See R C Speth. Sarcosine 11, glycine 8 angiotensin II is an AT1 angiotensin II receptor subtype selective anatagonist). Regulatory peptides 115 (2003) 203-209)
As mentioned above, the natural ligand to the AT1 receptor is the octapeptide AngII, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, which binds to the AT1 receptor in the nano-mole range.
When modifying a naturally binding ligand to a receptor by the binding of a moiety, in particularly with moieties that are relatively large and relatively bulky, the affinity of the peptide vector is frequently compromised.